Sustained inhibition of PKCα reduces intravasation and lung seeding during mammary tumor metastasis in an in vivo mouse model - PubMed (original) (raw)

Sustained inhibition of PKCα reduces intravasation and lung seeding during mammary tumor metastasis in an in vivo mouse model

J Kim et al. Oncogene. 2011.

Abstract

Metastasis is the major reason for breast cancer-related deaths. Although there is a host of indirect evidence for a role of protein kinase C (PKC) α in primary breast cancer growth, its role in the molecular pathways leading to metastasis has not been studied comprehensively. By treating mice with αV5-3, a novel peptide inhibitor selective for PKCα, we were able to determine how PKCα regulates metastasis of mammary cancer cells using a syngeneic and orthotopic model. The primary tumor growth was not affected by αV5-3 treatment. However, the mortality rate was reduced and metastasis in the lung decreased by more than 90% in the αV5-3-treated mice relative to the control-treated mice. αV5-3 treatment reduced intravasation by reducing matrix metalloproteinase-9 activities. αV5-3 treatment also reduced lung seeding of tumor cells and decreased cell migration, effects that were accompanied by a reduction in nuclear factor kappa B activity and cell surface levels of the CXCL12 receptor, CXCR4. αV5-3 treatment caused no apparent toxicity in non-tumor-bearing naïve mice. Rather, inhibiting PKCα protected against liver damage and increased the number of immune cells in tumor-bearing mice. Importantly, αV5-3 showed superior efficacy relative to anti-CXCR4 antibody in reducing metastasis in vivo. Together, these data show that pharmacological inhibition of PKCα effectively reduces mammary cancer metastasis by targeting intravasation and lung seeding steps in the metastatic process and suggest that PKCα-specific inhibitors, such as αV5-3, can be used to study the mechanistic roles of PKCα specifically and may provide a safe and effective treatment for the prevention of lung metastasis of breast cancer patients.

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Conflict of interest statement

Conflict of interest

DMR is the founder of KAI Pharmaceuticals, Inc. However, none of the work at her laboratory was supported by the company and the company had no access to information about unpublished research. Drs. Kim, Thorne, Huang and Mochly-Rosen and Lihan Sun declare no conflict of interest.

Figures

Figure 1

Figure 1. PKCα is more active in metastatic 4T1 mammary cancer cells relative to non-metastatic cancer cells

(A) 4T1 and JC cells were transfected with firefly luciferase and 100,000 cells were injected orthotopically into the mammary fat pad of 6 week old female BALB/c mice (n= 12 each). Primary tumor growth (Figure 1A, top) and lung metastases (bottom) of the two cell lines were compared in vivo by bioluminescence imaging using an IVIS100. 4T1-luc is shown in blue and JC-luc in red. Sample bioluminescence images of lung metastases are shown on right. (B) Subcellular distribution of PKCα, βII and ɛ between the cytosolic (C) and particulate (P) fractions (expressed as percent enzyme in the particulate fraction, a measure of activation of PKC) in 4T1-luc vs. JC-luc cells, was determined by Western blot analysis (IB) of cultured cell lysates. (n=3 each, *; p<0.05; NS for PKCɛ). (C) Subcellular distribution of PKCα, βII and ε between the cytosolic and particulate fractions in 4 week-old 4T1 tumors grown in BALB/c mice (n=4 each, *; p<0.05 vs. PKCα). Loading controls for cytosolic and particulate fractions (GAPDH and Gαi) are shown.

Figure 2

Figure 2. Inhibition of PKCα reduces tumor metastasis

(A) Experimental protocol: 4T1-luc tumor cells were injected (100, 000/0.1mL) into the mammary fat pad of BALB/c female mice (n=6–12). One week after cell injection, treatment with peptides was started using Alzet mini pumps for 4 weeks and mice were subsequently sacrificed. Treatment with αV5-3 for 4 weeks significantly reduced metastasis to lungs (B, top) and rib cages (B, bottom *; p<0.05, unpaired t test). (C) Treatment with αV5-3 decreased active levels of PKCα in the tumors as measured by translocation assay. GAPDH and Gαi are used as loading controls for cytosolic (C) and particulate (P) fractions, respectively. (D) αV5-3 treatment did not affect the active level of PKCβII as measured by translocation assay (Figure 2D, n=4 each, *; p<0.05, unpaired t test). Same loading controls were used for Figures 2C and D.

Figure 3

Figure 3. Inhibition of PKCα blocks metastasis at the intravasation stage

(A) Intravasation of 4T1-luc cells through a mouse endothelial cell layer and into a matrigel layer was measured using bioluminescence imaging (IVIS50, Xenogen, part of Caliper Life Science, n=4). Mouse tumor endothelial cells (2H-11, ATCC) were grown on top of a Matrigel plug in tissue culture inserts in 24-well plates until they formed a confluent monolayer. Breast cancer cells (1,000,000 cells/well) expressing luciferase were then added above the endothelial cell layer and peptides were added as indicated, to a final concentration of 10µM. Peptides were re-applied every 2h for 10 hours and the cells were then incubated for a further 14 hours (a total of 24h). At the end of this time, cell media was aspirated and a cotton swab was used to remove the endothelial cell layer. The matrigel plug was then imaged (IVIS50; Xenogen, part of Caliper Life Sciences) after addition of luciferin. Bioluminescence produced was used to quantify the number of labeled tumor cells that had crossed the endothelial cell layer and entered the matrigel plug. (B) Experiment was repeated looking at invasion of human MDA-MB-231-luc breast cancer cells across primary human endothelial HUVEC cells (TAT treatment in clear bars and αV5-3 treatment in blue bars, n=4). (C) Activities of MMP-9 in primary tumors were measured by in-gel zymography in homogenates of tumors isolated from mice treated as described in Figure 2. Molecular weights of pro- and active-forms of MMP-9 are shown. (D) Activities of secreted MMP-9 were measured from cultures of 4T1-luc cells treated with vehicle (DMSO, control) and SB-3CT (an MMP-9 inhibitor, 10µM in DMSO) (n=3 for each). Cells were treated for 24 hours and media was collected and analyzed for MMP-9 activities. Also, MMP-9 activities were measured in the medium from cells treated with control (ctrl) siRNA and siRNA of PKCα for 48 hours and cultured for 2 more days.

Figure 4

Figure 4. Inhibition of PKCα protects against lung seeding of cancer cells

(A) 4T1-luc tumor cells were injected via the tail vein (100,000 cells/0.1mL PBS, n=9–10 each). Administration of peptides by osmotic pumps was begun 2 days before the tumor cell injection. Animals were then imaged on day 5 post-tumor cell injection to measure the extent of lung seeding and metastasis. (B) Lungs from this study were recovered 14 days post-treatment, stained with Hematoxylin to identify number and size of 4T1-tumor nodules (n=3 each, representative images are shown at 200X; arrows indicate tumor nodules, scale bar; 10µm). (C) Comparison of survival rate between TAT-treated and αV5-3-treated groups. Animals were monitored up to 30 days after the tumor cell injection for survival analysis as plotted on Kaplan-Meier survival curves (n=4–6, each).

Figure 5

Figure 5. αV5-3 reduces cell migration, reduces cell surface levels of CXCR4 and produces a greater reduction in metastasis than anti-CXCR4 antibody in vivo

(A) 4T1 Cells (2.5×104 in 0.5ml of media) were serum starved and were pre-treated for one day every 3 hours for 9 hours with TAT or αV5-3 at 1µM. On the day of the experiment, the inserts were placed in wells with serum containing (10% FBS) media. The same number of control inserts was placed in empty wells of the BD companion plates. Cells were added on top of the wells and incubated with TAT or αV5-3 at 1µM for 24 hours. After incubation, cells migrated (to control inserts) and invaded (to matrigel inserts) were counted and percentage of cells invaded/migrated was calculated for each group. (B) Primary tumor lysates from animals treated as in Figure 2 were isolated, dissociated, stained with anti-CXCR4-FITC antibody and were analyzed by flow cytometry to measure the cell surface levels of CXCR4 (n=4 each). (C) Tumors from mice treated with TAT or αV5-3 for 4 weeks were analyzed for phospho-IκBα and non-phosphorylated IκBα levels (n=4 each). The ratio of phospho-IκBα to IκBα is shown. GAPDH was used as a loading control. (D) Comparison of the anti-metastatic effects of αV5-3 with PDTC (NF-κB inhibitor) or anti CXCR4 antibody. Peptides were dissolved in saline and administered at a constant rate (0.5µl/hr) corresponding to 24 mg/kg/day (30mM TAT) and 36 mg/kg/day (30mM αV5-3-TAT conjugate or αV5-3, in short). Pyrrolidine dithiocarbamate (PDTC, Sigma, P-8765, 50 mg/kg/day) or anti-CXCR4 antibody (10mg/ml) was also delivered in osmotic pumps. Bioluminescence imaging (BLI) was used to measure extent of upper abdomen metastasis after 14 days (n=5–7 each, *; p<0.05 vs. TAT). (E) Primary tumor lysates from animals treated as in (D) were isolated, dissociated, stained with anti-CXCR4-FITC antibody and were analyzed by flow cytometry to measure the cell surface levels of CXCR4 (n=5–7 each).

Figure 6

Figure 6. Summary of PKCα-mediated metastatic pathways in mammary cancer cells

A scheme summarizing the PKCα-mediated mechanisms in metastasis. A summary of potential sites of action of PKCα during metastatic processes is shown. PKCα can be activated by various factors, including activation of ErbB2 or G protein-coupled receptor (GPCR) or growth factors, etc. PKCα regulates intravasation via MMP-9 activity and cell surface CXCR4 levels, inducing higher migration and possibly more survival of cancer cells. Lung seeding and metastatic spread in the lungs and other secondary sites are also regulated by active PKCα. Inhibitory peptide against PKCα, αV5-3, inhibited intravasation, migration and lung seeding/metastases of mammary cancer cells in vivo. Green cells: tumor cells, yellow cells: neutrophils, dark pink cells: macrophages, coiled receptor: CXCR4 and blue hexagon: CXCL12. Active PKCα is shown in red.

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